This proposal will address a novel aspect of ethanol's developmental neurotoxicity, i.e. its effect on cholesterol homeostasis in the central nervous system. While too much cholesterol may be deleterious, as in case of atherosclerosis and Alzheimer's disease, too little can produce birth defects. Different degrees of mental retardation are often observed in inborn errors of cholesterol synthesis such as Smith-Lemli-Opitz syndrome. Lack of cholesterol during brain development as a consequence of these genetic defects leads to severe brain damage, including microcephaly and mental retardation, both of which are hallmarks of the fetal alcohol syndrome. The effects of ethanol on cholesterol homeostasis in the developing brain have not been investigated. In the brain, cholesterol is mostly produced endogenously, and its homeostasis is regulated by endogenously produced lipoproteins and cholesterol transporters. This proposal will investigate the hypothesis that ethanol, by upregulating cholesterol transporters and lipoprotein production by central nervous system cells, will increase the clearance of cholesterol from the brain, causing cholesterol depletion. Low levels of cholesterol are consistent with some of the effects caused by in utero alcohol exposure such as inhibition of the sonic hedgehog pathway, neuronal development and survival, and cell proliferation. Specific Aims of the proposal are: 1) The investigation of the role of ABCA1 and ABCG1 in ethanol-induced cholesterol efflux. The effect of ethanol on cholesterol efflux and cholesterol levels will be investigated in cortical neurons in vitro;in addition, the role of ABCA1, ABCG1, and phospholipase D in ethanol-induced upregulation of cholesterol efflux and cholesterol transporter induction in astrocytes and, possibly, in neurons will be examined by selectively removing these proteins form astrocyte and neuron cultures. 2) The investigation of the effect of ethanol on astrocyte-generated lipoproteins and their interaction with neurons. The effect of ethanol on the composition of astrocyte-produced lipoproteins separated by gel filtration, and their role on cholesterol efflux from neurons will be analyzed. Furthermore, the effect of ethanol on lipoprotein composition and cholesterol efflux will be examined in an astrocyte/neuron co- culture system. 3) The investigation of the effect of in vivo ethanol administration on ABC cholesterol transporters and on cholesterol levels in the developing brain. The effect of in vivo exposure to ethanol during gestation on the cholesterol transporters ABCA1 and ABCG1 transcription and expression in neurons and astrocytes and on cholesterol levels will be assessed in the neocortex of rat fetuses at gestational day 21. Altogether, these studies are directed at characterizing a possible novel mechanism involved in ethanol-induced neurodevelopmental effects, i.e. its effects on cholesterol homeostasis in the brain. PUBLIC HEALTH RELEVANCE The proposed studies on the effect of ethanol on cholesterol homeostasis in the developing brain were prompted by the observation that several of the neurodevelopmental effects caused by ethanol are consistent with effects caused by lack of cholesterol. Cholesterol is indeed necessary for various aspects of brain development. We hypothesized that ethanol, by mechanisms to be investigated in the proposed project, may affect cholesterol homoeostasis and reduce cholesterol levels in the developing brain. The most recent discoveries in the field of cholesterol trafficking will be applied to the investigation of the effects of ethanol on cholesterol transporters, lipoprotein generation and, ultimately, cholesterol levels in the developing brain. As cholesterol is a regular component of the diet, our studies could potentially lead to the revision of dietary guidelines for pregnant women at risk. It may also help understanding why in some very poor regions of South Africa the prevalence of FAS is much higher than in Western countries as these populations also experience severe malnutrition.